1. Field of the Invention
The present invention provides human conjunctival epithelial cell lines with an extended life span. These conjunctival cell lines can be cultured as stratified 3-dimensional (3-D) cultures. The 3-D cultures can be used as tissue- and species-specific cellular models of the human conjunctival ocular surface. These human conjunctival cultures are particularly useful in product safety evaluations to test products for their eye irritation potential.
2. Description of the Related Art
Injury to the eye from exposure to chemicals can result in blindness. Accordingly, to protect against inadvertent damage from commercially applied materials, the Food, Drug, and Cosmetic Act of 1938 has required testing of cosmetic and drug products.
Prior to the present invention, the most common method for testing ocular irritancy in humans employed rabbits. The test, known as the "Draize Test," involves placing the foreign substance to be tested directly into the conjunctival sac of the rabbit eye. The Draize Test was first described in Draize et al (1944) J. Pharm. Exp. Ther. 83:377-390. The Draize Test is simple to perform, provides quick, economical results, and uses a laboratory animal which is easy to breed and maintain. The conjunctiva and cornea provide different components of the overall biological response of the ocular surface, and in particular the cornea and conjunctiva, to noxious compounds.
The corneal epithelium is a transparent barrier that also aids in maintaining the transparency of the underlying corneal stroma. The transparency allows light penetration to the retina and is crucial to visual acuity. The corneal response in the Draize test is assessed by the degree of damage to the corneal epithelium, and the degree of corneal opacity, resulting from contact with the test substance. The cornea is highly innervated to within several microns of the ocular surface, and thus provides a significant part of the neural response (burning, stinging, etc.) to eye irritants.
The conjunctiva is the tissue that lines the eyelids, and covers the anterior portion of the globe of the eye (except for the cornea). The conjunctiva consists of a delicate membrane composed of an epithelium, and a substantia propria, that overlie the tough outer portion of the eyeball known as the sclera. The epithelium is stratified into multiple cell layers, and provides a barrier to the penetration of compounds into the eye. There are desmosomal junctions connecting the epithelial cells to one another, and tight junctions between the surface cells that prevent the penetration of small ions. Other cells that can be found in the normal conjunctival epithelium include: goblet cells, which are specialized cells that secrete mucin; Langerhans cells; melanocytes; a small population of immune cells (lymphocytes and neutrophils); and an interspersed neuronal component. Beneath the epithelium, the substantia propria contains stromal cells interspersed in a layer of connective tissue. The substantia propria also contains a microvasculature, lymphatics, immune cells, and neurons. Mast cells are not found in the normal conjunctival epithelium, but tissue type mast cells reside in the substantia propria. The conjunctiva responds to eye irritants by mounting an inflammatory response. The conjunctival response is assessed in the Draize rabbit eye test as redness, chemosis, and discharge.
However, there are drawbacks to the Draize Test, including the morphological differences between the rabbit eye and the human eye, overprediction of the Draize test, variability from animal to animal and from lab to lab, and the adamant opposition of animal rights activists to the use of animals in such tests.
In vitro models for human corneal epithelium have been developed, including those described in U.S. Pat. No. 5,672,498, U.S. Pat. No. 5,786,201, and U.S. Pat. No. 5,585,265, the contents of all of which are hereby incorporated by reference. However, toxicologists, regulatory agency scientists and ophthalmologists have questioned the validity of extrapolating results from an in vitro model that represented only corneal effects to the Draize test, which represents corneal (73%), conjunctival (18%), and iridial (9%) responses. The conjunctival response to noxious compounds has been identified as an important element of the human ocular response, and as independent of the corneal response for at least some compounds. Indeed, it has been noted that ocular exposures (Draize tests) to some products resulted in an adverse reaction in only the conjunctiva of the rabbit. Examples have also been reported in the literature where conjunctival signs were the only clinical response to an ocular exposure.
It is, therefore, thought that combined conjunctival and corneal response data will provide information more likely to be representative of the overall human ocular response from exposure to a noxious compound. Multiple tests that measure different mechanisms of injury in the two tissues provide a composite test battery that can serve as a replacement for the Draize test. With this type of test system, compounds that elicit primarily a conjunctival response will not be "missed" by testing on only a corneal model, thereby providing a more effective in vitro test scheme.
However, prior to the present invention, it has been extremely difficult to assess conjunctival response to exposure to noxious substances in vitro. There are no reports of transfected conjunctival cell lines, either human or animal, in the scientific literature. The only conjunctival cell line available from the American Type Culture Collection (ATCC) is the Chang cell line. Accordingly, investigators wishing to study the human conjunctiva must do clinical studies, or use primary conjunctival tissue or cell cultures. For in vitro studies three possible sources of conjunctival cells exist: (1) the Chang cell line; (2) primary human conjunctival cell cultures; and (3) primary human conjunctival tissue specimens.
The Chang conjunctival cell line is described to be of human conjunctival origin (Chang, 1954; Wong & Kilbourne, 1961). The Wong-Kilbourne derivative of Chang conjunctiva is available from the ATCC (ATCC CCL 20.2.; ATCC Catalog, 7th ed., 1992). There are two principal problems with using Chang cells. First, Chang cell cultures do not retain many of the characteristics of the conjunctival epithelium. No publication has been found that compares the characteristics of Chang cells and primary conjunctival epithelial cells. Most authors note that data obtained using the Chang cell line should be interpreted with caution. Reports citing the use of the Chang cell line are frequently for viral propagation studies, since it is not commonly used for studies on conjunctival biology. Second, the Chang cell line from the ATCC is reported to contain HeLa cell marker chromosomes. ATCC recommends that "such lines should not be chosen for study when the specific organ or tissue of presumptive origin is of importance to the validity of the research."
Many investigators use primary cultures of human conjunctival epithelial cells. Primary cells are most often cultured as monolayers, but have been cultured stratified on permeable membranes. Problems with using primary conjunctival cells include limited availability, biological variability, difficulty in establishing the cultures, potential contamination with infectious agents, finite life spans, and multiple cell types. Primary conjunctival cells and tissues are typically obtained from surgery specimens, limiting the amount available. Conjunctival tissue is highly vascularized, and degrades rapidly after donor death. It must be harvested within 15 hours, and preferably within 8 hours postmortem. Many eye banks cannot accommodate this type of tissue collection schedule, which highly limits access to fresh conjunctival tissue. Most investigators that use fresh conjunctival tissue are professionally associated with an eye bank. Conjunctival tissue can also be isolated and used as pieces of tissue, or maintained as an organ cultured tissue. However, the use of such tissue cultures presents the same problems as the use of primary cultures.
Other in vitro models that have been used for the evaluation of eye irritation include: monolayer cultures of animal or human ocular cells (cornea or conjunctival), or skin cells; monolayer cultures of MDCK cells on porous membranes (dog kidney epithelial cell line); multilayered cultures composed of human skin cells; a variety of other non-species and non-tissue specific in vitro models, including EYTEX, CAM, CAMVA, HET-CAM; multilayered cultures of primary, non-human ocular cells; and multilayered cultures of primary, human ocular cells. Problems with these in vitro models include:
1) The limited, or lack of, biological relevance to the tissue of the ocular surface. Monolayer cultures do not form the same barrier to toxic compounds that is found at the in vivo ocular surface. Monolayer cultures are more susceptible to chemical injury and do not recover as well as stratified tissue constructs (Ward, 1997). Non-human and non-ocular cell types are not biologically relevant for the assessment of human eye irritation. PA0 2) Appropriate endpoints of toxicity and irritation for the conjunctival ocular surface have not been determined. Non-ocular and non-human cells may not be useful for this determination. Monolayer cultures do not provide an adequate response that is translatable to the human ocular surface response to injury/irritation.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of evaluating the eye irritation potential of consumer products in vitro, it should be apparent that there still exists a need in the art for a continuous conjunctival epithelial cell line.